Amine oxidase mutant and application thereof in synthesis of chiral compound

Abstract

The invention discloses an amine oxidase mutant and application thereof in synthesis of chiral compounds, and relates to the field of biology. The amine oxidase mutant provided by the invention has mutation at any one or more sites of the 47 site, the 182 site, the 183 site, the 275 site, the 282 site and the 335 site of a wild type amino acid sequence, has relatively high catalytic activity on (S)-3-aminobutanol and relatively good stereoselectivity, can selectively oxidize (S)-3-aminobutanol under the condition that (R)-3-aminobutanol is not influenced, and has good application prospects. When the 4-hydroxy-2-butanone is applied to catalysis of (R)-3-aminobutanol feed liquid containing (S)-3-aminobutanol, the R-type ee value in the feed liquid can be effectively increased, additional refining and purifying steps are not needed, and the cost is remarkably reduced.

Description

Technical Field

[0001] This invention relates to the field of biology, and more specifically, to amine oxidase mutants and their application in the synthesis of chiral compounds. Background Technology

[0002] (R)-3-aminobutanol is a key chiral intermediate for the anti-AIDS drug dulutegravir, and its product quality plays a crucial role in the overall quality of dulutegravir. With the development of the pharmaceutical industry, the purity requirements for (R)-3-aminobutanol are becoming increasingly stringent, especially regarding ee value and impurity content.

[0003] Currently, both chemical and biocatalytic methods are used to synthesize high-purity (R)-3-aminobutanol. Biocatalysis, with its significant advantages of mild reaction conditions, low environmental pollution, and high economic efficiency, has become a hot topic in research and application. Various enzymes, such as transaminases and amine dehydrogenases, have been developed for the biosynthesis of (R)-3-aminobutanol. The transaminase route uses 4-hydroxy-2-butanone as a substrate, transferring the amino group from inexpensive amino donors, such as isopropylamine and alanine, to the carbonyl carbon of the substrate via a transamination reaction. This route achieves a product yield of up to 95%, but the ee value is only 99.8%, failing to meet the quality standard requirement of an isomer content of less than 0.03% (ee value 99.94%). Currently, a tartaric acid chemical resolution step is added after the catalytic process to improve the ee value. However, this method not only consumes a large amount of resolution reagents but also increases subsequent extraction and purification steps, raising production costs by approximately 30%.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide amine oxidase mutants and their application in the synthesis of chiral compounds.

[0006] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide an amine oxidase mutant having a mutation at any one or more of the following positions in the amino acid sequence of the wild-type amine oxidase: position 47, position 182, position 183, position 275, position 282, and position 335.

[0007] Secondly, embodiments of the present invention provide an isolated nucleic acid molecule that encodes the amine oxidase mutant described in the foregoing embodiments.

[0008] Thirdly, embodiments of the present invention provide a recombinant vector containing the isolated nucleic acid molecules described in the foregoing embodiments.

[0009] Fourthly, embodiments of the present invention provide a recombinant cell containing the isolated nucleic acid molecules described in the foregoing embodiments or the recombinant vector described in the foregoing embodiments.

[0010] Fifthly, embodiments of the present invention provide a catalyst comprising the amine oxidase mutant described in the foregoing embodiments.

[0011] In a sixth aspect, embodiments of the present invention provide a method for preparing the amine oxidase mutant as described in the foregoing embodiments, comprising: artificially synthesizing the amine oxidase mutant or culturing the recombinant cells described in the foregoing embodiments.

[0012] In a seventh aspect, embodiments of the present invention provide the use of amine oxidase mutants as described in the foregoing embodiments, or isolated nucleic acid molecules as described in the foregoing embodiments, or recombinant vectors as described in the foregoing embodiments, or recombinant cells as described in the foregoing embodiments, or catalysts as described in the foregoing embodiments, in the preparation or purification of (S)-3-aminobutanol or chiral compounds.

[0013] Eighthly, embodiments of the present invention provide a method for purifying (R)-3-aminobutanol, comprising the following steps: adding the amine oxidase mutant described in the foregoing embodiments, the recombinant cells described in the foregoing embodiments, or the catalyst described in the foregoing embodiments to a reaction system containing (S)-3-aminobutanol and (R)-3-aminobutanol to carry out a catalytic reaction.

[0014] The present invention has the following beneficial effects: The amine oxidase mutant optimized in this invention exhibits high catalytic activity and good stereoselectivity for (S)-3-aminobutanol. It can selectively oxidize (S)-3-aminobutanol to generate 4-hydroxy-2-butanone without affecting (R)-3-aminobutanol. When applied to catalyze (R)-3-aminobutanol solutions containing (S)-3-aminobutanol, it can effectively increase the R-type ee value in the solution in a short time without the need for additional purification steps. This reduces the use of a large amount of resolving reagents in existing tartaric acid chemical resolution methods, significantly lowering costs. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a flowchart of the enzyme catalysis technology for amine oxidase under CAT enzyme and O2 conditions; Figure 2This is a diagram illustrating the principle of color development. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0018] In this invention, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The amino acid sequence identity percentage alignment can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA.

[0019] An embodiment of the present invention provides an amine oxidase mutant, wherein the amine oxidase mutant has a mutation at any one or more of the following positions in the amino acid sequence of the wild-type amine oxidase: position 47, position 182, position 183, position 275, position 282, and position 335.

[0020] In an optional embodiment, the amine oxidase mutant has any of the following mutations or combinations thereof relative to the wild-type amine oxidase: N47A, T182S, L183V, M275Q, Q282S, and F335Y.

[0021] In an optional embodiment, the amine oxidase mutant has any of the following mutation combinations relative to the wild-type amine oxidase: T182S / N47A, T182S / L183V, T182S / L183VM275Q, T182S / L183V / F335Y, T182S / L183V / Q282S, and T182S / L183V / Q282S / F335Y.

[0022] In an optional embodiment, the wild-type amine oxidase is derived from Aspergillus date palmatum (…). Aspergillus phoenicis ) amine oxidase.

[0023] In an optional embodiment, the amino acid sequence of the wild-type amine oxidase has at least 80% identity with the sequence shown in SEQ ID NO:1.

[0024] In an optional embodiment, the at least 80% includes 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or more, or 100%.

[0025] On the other hand, embodiments of the present invention provide an isolated nucleic acid molecule that encodes the amine oxidase mutant described in any of the foregoing embodiments.

[0026] On the other hand, embodiments of the present invention provide a recombinant vector containing the isolated nucleic acid molecules described in any of the foregoing embodiments.

[0027] In optional embodiments, the recombinant vector is an expression vector or a cloning vector, optionally an expression vector, which can refer to any recombinant polynucleotide construct. This construct can introduce the target DNA fragment directly or indirectly (e.g., packaged as a virus) into host cells via transformation, transfection, or transduction to express the target gene. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can ligate the target DNA fragment into the plasmid circle. Another type of vector is a viral vector, which can ligate and package the target DNA fragment into a viral genome (e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, they can express the target gene.

[0028] On the other hand, embodiments of the present invention provide a recombinant cell containing a nucleic acid molecule of any of the molecules described in any of the foregoing embodiments or a recombinant vector of any of the foregoing embodiments.

[0029] In optional embodiments, the recombinant cells can be prokaryotic cells, eukaryotic cells, or bacteriophages. The aforementioned prokaryotic cells include, but are not limited to, *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*. The aforementioned eukaryotic cells include fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*; insect cells such as *Ardisia crenata*; mammalian cells such as BHK cells, CHO cells, COS cells, NSO cells, 293 series cells, HepG2, HEK293 cell lines, Huh7 cells, and myeloma cells, but do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0030] In an optional embodiment, the recombinant cells comprise recombinant bacteria. The recombinant bacteria can be prokaryotic or eukaryotic recombinant bacteria. The prokaryotic recombinant bacteria include, but are not limited to, *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*. The eukaryotic recombinant bacteria include, but are not limited to, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharizoa*, *Trichoderma*, and other fungi.

[0031] On the other hand, embodiments of the present invention also provide a catalyst containing the amine oxidase mutant described in any of the foregoing embodiments.

[0032] In an optional embodiment, the catalyst comprises: recombinant cells or cultures thereof as described in any of the foregoing embodiments.

[0033] In an optional embodiment, the preparation of the recombinant cell culture includes: S1: culturing the recombinant cells to OD. 600 S2: Add isopropyl-β-D-thiogalactoside to a concentration of 0.01-1 mM and incubate at 20-30℃ for 20-30 h; S3: Centrifuge, discard the supernatant, and collect the precipitate.

[0034] In an optional embodiment, the culture temperature in step S1 can be 30~45℃, or optionally 36~38℃.

[0035] In an optional embodiment, in step S2, the concentration of IPTG is 0.05~0.5mM, optionally 0.05~0.2mM.

[0036] On the other hand, embodiments of the present invention provide a method for preparing amine oxidase mutants as described in any of the foregoing embodiments, comprising: artificially synthesizing the amine oxidase mutants or culturing the recombinant cells described in any of the foregoing embodiments.

[0037] In optional embodiments, the culturing steps are the same as those described in the preparation of recombinant cell cultures in any of the foregoing embodiments, and will not be repeated here. On the other hand, embodiments of the present invention provide the application of amine oxidase mutants, isolated nucleic acid molecules, recombinant vectors, recombinant cells, or catalysts as described in any of the foregoing embodiments in the selective oxidation of (S)-3-aminobutanol or chiral compounds in their preparation or purification.

[0038] In an optional embodiment, the chiral compound includes (R)-3-aminobutanol.

[0039] On the other hand, embodiments of the present invention provide a method for preparing or purifying (R)-3-aminobutanol, which includes the following steps: adding the amine oxidase mutant described in any of the foregoing embodiments, the recombinant cell described in any of the foregoing embodiments, or the catalyst described in any of the foregoing embodiments to a reaction system containing (S)-3-aminobutanol and (R)-3-aminobutanol to carry out a catalytic reaction.

[0040] In an optional embodiment, the reaction system containing (S)-3-aminobutanol and (R)-3-aminobutanol is a reaction system containing R-3-aminobutanol solution.

[0041] In an optional embodiment, the (R)-3-aminobutanol solution comprises: (R)-3-aminobutanol solution produced by transaminase using 4-hydroxy-2-butanone as a substrate.

[0042] In an optional embodiment, the conditions for the catalytic reaction include: 20–40°C, 1–30 h, and 0.1–2 VVM. The temperature can be any one or a range between any two of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40°C; the time can be any one or a range between any two of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30 h; and the aeration rate can be any one or a range between any two of 0.1, 0.5, 1, 1.5, and 2 VVM.

[0043] In an optional embodiment, for every 0.05-0.5g of (S)-3-aminobutanol, 0.1-5g of the recombinant cells or their culture is added; specifically, 0.05-0.5g can be any one or any two of 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, and 0.5g; and 0.1-5g can be any one or any two of 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, and 5g.

[0044] In an optional embodiment, the reaction system further includes NAD oxidase or catalase (CAT enzyme) and a buffer solution.

[0045] Using (R)-3-aminobutanol as a substrate, a catalytic reaction was carried out under CAT enzyme and O2 conditions. The (S)-3-aminobutanol in the catalytic solution was converted to 4-hydroxy-2-butanone under the catalysis of amine oxidase. The enzyme catalytic pathway is shown in [Figure number missing]. Figure 1 .

[0046] In an optional embodiment, the catalase activity in the reaction system is 50-1000 U / L, and the concentration can be any one or any two of 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 U / L.

[0047] In an optional embodiment, the pH of the buffer solution is 7.4 to 7.8, specifically any one or any two of 7.4, 7.5, 7.6, 7.7 and 7.8.

[0048] In an optional embodiment, the buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer. The concentration of potassium dihydrogen phosphate-dipotassium hydrogen phosphate can be 40~60 mM, specifically any one or any two of 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 and 60 mM. The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0049] Example 1: Feasibility of wild-type amine oxidase catalysis of (S)-3-aminobutanol The enzymatic catalytic technology route is as follows: In the (R)-3-aminobutanol catalytic solution obtained by transaminase catalysis, the ee value of R-type 3-aminobutanol is often lower than 99.94%, failing to meet quality standards. This invention uses a mutant strain of amine oxidase (MAON) derived from *Aspergillus sphoenicis* as a catalyst, and the (R)-3-aminobutanol catalytic solution as a substrate. The catalytic reaction is carried out under CAT enzyme and O2 conditions. Ultimately, the S-type 3-aminobutanol in the catalytic solution is converted to 4-hydroxy-2-butanone under the catalysis of amine oxidase, thereby increasing the ee value of R-type 3-aminobutanol in the (R)-3-aminobutanol catalytic solution.

[0050] The article "Directed Evolution of an Amine Oxidase Possessing both Broad Substrate Specificity and High Enantioselectivity" describes a method catalyzed by an amine oxidase (SEQ ID NO: 1) to convert racemic mixtures into single R enantiomers. The enzyme gene used in the literature was synthesized, and a bacterial strain was constructed. The enzymatic catalytic effect on racemic 3-aminobutanol was then determined.

[0051] Amino acid sequence of amine oxidase (SEQ ID NO:1): MTSRDGYQWTPETGLTQGVPSLGVISPPTNIEDTDKDGPWDVIVIGGGYCGLTATRDLTVAGFKTLLLEARDRIGGRSWSSNIDGYPYEMGGTWVHWHQSHVWREITRYKMHNALSSPFNFSR GVNHFQLRTNPTTSTYMTHEAEDELLRSALHKFTNVDGTNGRTVLPFPHDMFYVPEFRKYDEMSYSERIDQIRDELSLNERSSLEAFILLCSGGTLENSSFGEFLHWWAMSGYTYQGCMDCLIS YKFKDGQSAFARRFWEEAAGTGRLGYVFGCPVRSVVNERDAARVTARDGREFVAKRVVCTIPLNVLSTIQFSPALSTERISAMQAGHVNMCTKVHAEVDNMDMRSWTGIAYPFNKLCYAIGDGT TPAGNTHLVCFGTDANHIQPDEDVRETLKAVGQLAPGTFGVKRLVFHNWVKDEFAKGAWFFSRPGMVSECLQGLREKHGGVVFANSDWALGWRSFIDGAIEEGTRAARVVLEELGTKREVKARL

[0052] First, codon optimization was performed on *E. coli* based on the amino acid sequence of amine oxidase derived from *Aspergillus phoenicis*. Then, the nucleic acid fragment encoding the gene was chemically synthesized and ligated into the plasmid vector pET-28a(+) via NcoI and XhoI restriction sites. Finally, the plasmid was introduced into *E. coli* BL21(DE3) using CaCl2-mediated chemical transformation. The resulting single-colony transformants were the recombinant expression strain of amine oxidase.

[0053] The recombinant strain was inoculated at a 1% inoculum into 5 mL of LB medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm for 15 h to obtain primary seed culture. Then, the primary seed culture was transferred at a 4% inoculum into 50 mL of TB medium containing 50 mg / L kanamycin and cultured at 37°C and 220 rpm until the OD value of the bacterial culture reached 0.6–0.8. IPTG at a final concentration of 0.1 mM was then added to induce expression at 25°C and 220 rpm for 24 h. After fermentation, the supernatant was removed by centrifugation at 4000 rpm for 15 min, and the MAON amine oxidase cells were collected.

[0054] The validation system consisted of 10 g / L racemic 3-aminobutanol, 10 g / L MAON amine oxidase cells, and 1000 U / L CAT enzyme in 100 mL of 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate (pH 7.6) buffer; the aeration rate was set to 1 VVM; and catalysis was performed at 30 °C for 24 h.

[0055] After the reaction, samples were taken to detect the changes in the content of (S)-3-aminobutanol and (R)-3-aminobutanol. The results showed that the mass of (S)-3-aminobutanol decreased by 61%, while that of (R)-3-aminobutanol decreased by only 8%. The R-type ee value of the racemic 3-aminobutanol solution increased from 0% to 40.5%, indicating that amine oxidase has the function of increasing the R-type ee value of the 3-aminobutanol solution. However, its activity and stereoselectivity are poor, and it cannot completely convert (S)-3-aminobutanol. Furthermore, it consumes the target analyte (R)-3-aminobutanol.

[0056] Example 2: Enhancing the activity and stereoselectivity of amine oxidases through enzyme molecule modification Based on wild-type amine oxidase (amino acid sequence as shown in SEQ ID NO:1), saturated mutant libraries were constructed by selecting G22, I25, S26, E45, N47, Y68, F72, T182, L183, Q217, P246, A274, M275, P279, Q282, I283, Y305, K307, F335, L337, F352, and W396 sites, and high-throughput screening was performed. After secondary screening of mutant strains, beneficial mutant strains N47A, T182S, L183V, M275Q, Q282S, and F335Y were obtained, showing enhanced activity and stereoselectivity. Among them, T182S showed the highest activity increase (2.02-fold). Using T182S as a template, random combinations of other sites were carried out, ultimately yielding the mutant strain T182S / L183V / F335Y with the best activity and selectivity. After 4 h of catalysis with racemic 3-aminobutanol, (S)-3-aminobutanol was 99.99% converted, while (R)-3-aminobutanol did not decrease, and the ee value increased to 99.98%.

[0057] The specific method is as follows: (1) Primary screening: saturation mutagenesis, 96-well plate fermentation and high-throughput screening Using MAON plasmid DNA as a template, PCR amplification was performed using PrimeStarMax enzyme. The PCR reaction volume was 50 μL: containing 25 μL PrimeStarMax enzyme, 2 μL each of forward and reverse primers, 1 μL template DNA, and the remainder made up with pure water. The reaction program was as follows: 98℃ pre-denaturation for 5 min; followed by 98℃ denaturation for 30 s, 58℃ annealing for 15 s, and 72℃ extension for 1 min 10 s, for a total of 30 cycles; and a final extension at 72℃ for 10 min.

[0058] A fragment product of approximately 6000 bp was amplified, digested with DpnI enzyme, and transformed into *E. coli* BL21(DE3) competent cells. The cells were plated and incubated overnight at 37°C. 200 μL of LB liquid medium containing 50 mg / L kanamycin was added to each well of a 96-well plate. Single colonies were picked from the transformation plates using a sterile toothpick and inoculated. The culture was incubated overnight at 37°C and 250 rpm to obtain the primary seed culture. 40 μL of the primary seed culture was transferred to TB liquid medium containing 50 mg / L kanamycin and incubated at 37°C until OD500. 600 When the pH reaches 0.6-0.8, IPTG at a final concentration of 0.1 mM is added for induction, and the mixture is incubated overnight at 25°C. 20 μL of the fermentation broth is lysed with lysozyme and added to 200 μL of the chromogenic solution for high-resolution sieving. The chromogenic solution is prepared as a 50 mM potassium dihydrogen phosphate-dipoxetine (pH 7.6) buffer solution, containing 10 g / L racemic 3-aminobutanol, 1.5 U / L horseradish peroxidase, 6 mM 4-aminoantipyrine, and 16 mM 2,4,6,tribromo-3-hydroxybenzoic acid. After reacting at 30°C for 30 min, the absorbance at 510 nm is measured using a microplate reader.

[0059] Colorimetric principle: 4-Aminoantipyrine (4-AAP, M=203.24) reacts with 2,4,6-tribromo-3-hydroxybenzoic acid (TBHBA, M=374.81) under the action of hydrogen peroxide to form a quinone compound, which appears red and has a characteristic peak around 510 nm. Figure 2 .

[0060] (2) Secondary screening Strains with colorimetric values ​​20% higher than the control strains were re-screened. The re-screening system consisted of a 50 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphate (pH 7.6) buffer solution, 10 g / L racemic-3-aminobutanol, 10 g / L MAON amine oxidase cells, 1000 U / L CAT enzyme, an aeration rate of 1 VVM, and catalysis at 30°C for 4 h. The content of aminobutanol and the ee value in the reaction solution were then measured.

[0061] Enzyme activity definition: Under the above reaction conditions, the amount of enzyme required to catalyze the conversion of 1 μmol (S)-3-aminobutanol per minute is defined as 1 enzyme activity unit (U); unit enzyme activity refers to the enzyme activity units (U / g) contained in each gram of wet cells; the relative enzyme activity of wild-type MAON is defined as 100%. Enantioselectivity definition: In aminobutanol catalyzed by amine oxidase oxidation, the difference between (S)-3-aminobutanol and (R)-3-aminobutanol / the sum of (S)-3-aminobutanol and (R)-3-aminobutanol × 100%.

[0062] Mutant strains with significantly enhanced enzyme activity and selectivity were identified by sequencing to determine the mutation type. The results are shown in Table 1.

[0063] Table 1 Single-point mutation

[0064] (3) Mutant combination The MAON mutant 2 (T182S) showed the highest activity increase (2.02-fold). Using T182S as a template, random combinations of other sites were carried out, and the activity and selectivity of the mutant strains were measured. The mutant strain 10 (T182S / L183V / F335Y) was obtained by screening and showed the highest increase in enzyme activity and selectivity.

[0065] Table 2 Mutation Combinations

[0066] Example 3: Effect of the optimal mutant strain on R-3-aminobutanol solution The method for producing transaminase cells used in this embodiment is the same as in Embodiment 1. (1) 500 g of potassium phosphate buffer solution (50 mM, pH 7.0) and 72 g of isopropylamine were added to a 2 L reactor. A 50% sulfuric acid solution was added to adjust the pH to 7.6. Then, 50 g of butanone (4-hydroxy-2-butanone), 5 g of transaminase-containing wet cells, and 0.25 g of PLP were added. The buffer solution was then added to bring the total volume to 1000 g. The reaction was carried out at 40 °C and 250 rpm for 18 h. After the reaction was completed, (R)-3-aminobutanol was obtained. The concentration of aminobutanol in the product was 48.2 g / L, and the ee value (R type) was 99.82%.

[0068] The (R)-3-aminobutanol solution was treated with the optimal amine oxidase mutant strain (mutant strain 10 (T182S / L183V / F335Y)). The (S)-3-aminobutanol content in the solution was only 0.05 g / L. The amount of mutant strain 10 was 1 g / L (wet cells), the amount of CAT enzyme was 100 U / L, the aeration rate was 1 VVM, the temperature was 30℃, and after catalytic reaction for 2 h, the contents of (R)-3-aminobutanol and (S)-3-aminobutanol were measured. The ee value (R type) was calculated to increase from 99.82% to 99.98%. (2) 500 g of potassium phosphate buffer solution (50 mM, pH 7.0) and 86 g of isopropylamine were added to a 2 L reactor. A 50% sulfuric acid solution was added to adjust the pH to 7.6. Then, 60 g of butanone alcohol, 5 g of wet transaminase cells, and 0.25 g of PLP were added. The buffer solution was then added to bring the total volume to 1000 g. The reaction was carried out at 40 °C and 250 rpm for 18 h. After the reaction was completed, (R)-3-aminobutanol was obtained. The concentration of aminobutanol in the product was 57.6 g / L, and the ee value (R-type) was 99.74%.

[0070] The (R)-3-aminobutanol solution was treated with the optimal amine oxidase mutant strain (mutant strain 10 (T182S / L183V / F335Y)). The (S)-3-aminobutanol content in the solution was only 0.09 g / L. The amount of mutant strain 10 was 1 g / L (wet cells), the amount of CAT enzyme was 100 U / L, the aeration rate was 1 VVM, the temperature was 30℃, and after catalytic reaction for 2 h, the contents of (R)-3-aminobutanol and (S)-3-aminobutanol were measured. The calculated ee value (R type) increased from 99.74% to 99.97%. (3) 500 g of potassium phosphate buffer solution (50 mM, pH 7.0) and 100 g of isopropylamine were added to a 2 L reactor. A 50% sulfuric acid solution was added to adjust the pH to 7.6. Then, 70 g of butanone alcohol, 7 g of transaminase wet cells, and 0.25 g of PLP were added. The buffer solution was then added to bring the total volume to 1000 g. The reaction was carried out at 40 °C and 250 rpm for 18 h. After the reaction was completed, (R)-3-aminobutanol was obtained. The concentration of aminobutanol in the product was 67.1 g / L, and the ee value (R type) was 99.72%.

[0072] The (R)-3-aminobutanol solution was treated with the optimal amine oxidase mutant strain (mutant strain 10 (T182S / L183V / F335Y)). The (S)-3-aminobutanol content in the solution was only 0.10 g / L. The amount of mutant strain 10 was 1.5 g / L (wet cells), the CAT enzyme dosage was 150 U / L, the aeration rate remained at 1 VVM, the temperature was 30℃, and after catalytic reaction for 2 h, the contents of (R)-3-aminobutanol and (S)-3-aminobutanol were measured. The calculated ee value (R type) increased from 99.72% to 99.97%. (4) 500 g of potassium phosphate buffer solution (50 mM, pH 7.0), 128 g of isopropylamine, and 50% sulfuric acid solution were added to a 2 L reactor to adjust the pH to 7.6. Then, 90 g of butanone alcohol, 10 g of transaminase wet cells, and 0.25 g of PLP were added, and buffer solution was added to bring the total volume to 1000 g. The reaction was carried out at 40 °C and 250 rpm for 18 h. After the reaction was completed, (R)-3-aminobutanol was obtained. The concentration of aminobutanol in the product was 86.2 g / L, and the ee value (R type) was 99.04%.

[0074] The (R)-3-aminobutanol solution was treated with the optimal amine oxidase mutant strain (mutant strain 10 (T182S / L183V / F335Y)). The (S)-3-aminobutanol content in the solution was only 0.43 g / L. The amount of mutant strain 10 was 3 g / L (wet cells), the amount of CAT enzyme was 300 U / L, the aeration rate was 1 VVM, the temperature was 30℃, and after catalytic reaction for 2 h, the contents of (R)-3-aminobutanol and (S)-3-aminobutanol were measured. The calculated ee value (R type) increased from 99.04% to 99.96%.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An amine oxidase mutant, characterized in that, The amine oxidase mutant has a mutation at any one or more of the following positions in the amino acid sequence of the wild-type amine oxidase: position 47, position 182, position 183, position 275, position 282, and position 335.

2. The amine oxidase mutant according to claim 1, characterized in that, The amine oxidase mutant has any one of the following mutations or combinations thereof relative to the wild-type amine oxidase: N47A, T182S, L183V, M275Q, Q282S, and F335Y.

3. The amine oxidase mutant according to claim 2, characterized in that, The amine oxidase mutant has any of the following mutation combinations relative to the wild-type amine oxidase: T182S / N47A, T182S / L183V, T182S / L183V / M275Q, T182S / L183V / F335Y, T182S / L183V / Q282S, and T182S / L183V / Q282S / F335Y; Optionally, the amino acid sequence of the wild-type amine oxidase has at least 80% identity with the sequence shown in SEQ ID NO:

1.

4. An isolated nucleic acid molecule, characterized in that, It encodes the amine oxidase mutant according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that, It contains the isolated nucleic acid molecules as described in claim 4.

6. A recombinant cell, characterized in that, It contains the isolated nucleic acid molecule as described in claim 4 or the recombinant vector as described in claim 5; Optionally, the recombinant cells include recombinant bacteria.

7. A catalyst, characterized in that, It contains the amine oxidase mutant according to any one of claims 1 to 3; Optionally, the catalyst comprises: the recombinant cells or cultures thereof as described in claim 6.

8. The method for preparing the amine oxidase mutant according to any one of claims 1 to 3, characterized in that, It includes: The amine oxidase mutant is artificially synthesized or the recombinant cells of claim 6 are cultured.

9. The use of the amine oxidase mutant according to any one of claims 1 to 3, the isolated nucleic acid molecule according to claim 4, the recombinant vector according to claim 5, the recombinant cell according to claim 6, or the catalyst according to claim 7 in the selective oxidation of (S)-3-aminobutanol or chiral compounds in preparation or purification; Optionally, the chiral compound includes (R)-3-aminobutanol.

10. A method for purifying (R)-3-aminobutanol, characterized in that, It includes the following steps: The amine oxidase mutant of any one of claims 1 to 3, the recombinant cell of claim 6, or the catalyst of claim 7 is added to the reaction system containing (S)-3-aminobutanol and (R)-3-aminobutanol to carry out the catalytic reaction; Optionally, the reaction system containing (S)-3-aminobutanol and (R)-3-aminobutanol is a reaction system containing (R)-3-aminobutanol feed solution; Optionally, the (R)-3-aminobutanol solution comprises: (R)-3-aminobutanol solution produced by transaminase using 4-hydroxy-2-butanone as a substrate; Optionally, the conditions for the catalytic reaction include: 20~40℃, 1~30 h, 0.1~2 VVM; Optionally, for every 0.05-0.5g of (S)-3-aminobutanol, 0.1-5g of the recombinant cells or their culture is added to the reaction system; Optionally, the reaction system further includes: NAD oxidase or catalase, and / or buffer solution; Optionally, the catalase activity in the reaction system is 50~1000 U / L; Optionally, the pH of the buffer solution is 7.4 to 7.8; Optionally, the buffer solution is a potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer solution.

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