CN112522228B - R-aminotransferase from pseudomonas ammoxidation and synthesis method thereof - Google Patents

Abstract

The invention discloses an R-aminotransferase from pseudomonas ammoxidation, which is named PamAT, the nucleotide sequence of the gene is shown as SEQ NO.1, the amino acid sequence is shown as SEQ NO.2, the R-aminotransferase contains the amino acid sequence shown as SEQ NO.2 or has at least 90 percent of identity with SEQ NO.2 or is substituted, deleted or added with one or more amino acids, and a synthesis method thereof; by highly stereoselective is meant that one stereoisomer is present in an amount of at least about 1.1 times the other, the R-aminotransferase of the invention is highly stereoselective and has a broad substrate spectrum with great potential for use in the biological manufacture of chiral amines and unnatural amino acids.

Description

An R-aminase derived from ammonia-oxidizing Pseudonocardiomas and its synthesis method

Technical field

The invention relates to the field of biocatalysis, and mainly relates to a preparation method of R-aminase derived from Psendonocardia ammonioxydans and its application in the production of chiral amines and non-natural amino acids.

Background technique

Unnatural amino acids and chiral amines are key intermediates or key chiral modules in the synthesis of many fine chemicals and drugs, such as: sitagliptin, paclitaxel, cisplatin, glufosinate; in addition, peptides containing unnatural amino acids It is more stable than natural peptides and still maintains its biological activity in the presence of proteases. Therefore, unnatural amino acids will play an irreplaceable role in the development of new drugs, antibodies and artificial proteins that are highly stable and will not be rejected by the human body. . However, the difference between unnatural amino acids and natural amino acids is that existing chiral amines and unnatural amino acids are all synthesized organically, which has the disadvantages of high pressure, high temperature and high energy consumption. Moreover, organic synthesis is difficult to obtain optically pure products, and cannot be industrialized through fermentation. Production is contrary to the current concept of green and sustainable development.

Aminotransferases belong to the class of transferases and are usually used to catalyze the transfer of amino groups from amino donor compounds to amino acceptor compounds. Transaminase enzymes are commonly found in animals, plant tissues and microorganisms. According to the position of the amino group of its substrate, transaminase can be divided into α-aminase and ω-aminase: α-aminase is more common and can only catalyze the transfer of α-amino acids, while ω-aminase is rare and can not only catalyze the transfer of α-amino acid, but also It can catalyze the transfer of non-α amino groups, has a broader substrate spectrum and strict stereoselectivity, and can resolve amine racemates under mild conditions, or catalyze the asymmetric amination of the carbonyl group of prochiral substrates to produce chiral amines and unnatural amino acids. Transaminase is also involved in many metabolic pathways, including vitamin synthesis, carbon and nitrogen absorption, secondary metabolism, etc. Transaminase is a coenzyme 5'-pyridoxal phosphate (PLP)-dependent enzyme, that is, type IV transaminase, and its catalytic reaction is reversible. , during the catalytic process, PLP and 5'-pyrididamine phosphate (PMP) are converted to each other. In short, using R-aminase to produce optically pure chiral amines and non-natural amino acids can avoid the problems of high temperature and pressure, high energy consumption and high pollution in traditional chemical processes. Therefore, the present invention discloses a method of producing optically pure chiral amines and non-natural amino acids derived from ammonia oxidation. The R-aminotransferase of Mononas sp. and its application in the production of chiral amines and unnatural amino acids. The R-aminotransferase is named PamAT. It is a type IV aminotransferase with (R) stereoselectivity. The specific aminotransferase obtained by the present invention Stereoselective enzymes are the key to distinguishing them from chemical catalysts. They are in line with the concept of green production and are conducive to subsequent research and transformation and the promotion of industrial applications.

Contents of the invention

The invention discloses an R-aminase derived from ammonia-oxidizing bacterium Psendonocardiaammonioxydans and its application in catalyzing the production of chiral amines and unnatural amino acids.

In order to achieve the above object, the present invention provides an R-aminase derived from Psendonocardia ammonioxydans, naming the enzyme PamAT, or its modifications, functional equivalents, functional fragments or variants, It is characterized in that the amino acid sequence of the R-R-aminase includes a sequence selected from one of the following sequences: (1) the amino acid sequence shown in SEQ NO. 2; (2) the amino acid sequence shown has at least 90% identity. and an amino acid sequence with aminotransferase activity with highly stereoselective R-configuration catalytic activity; (3) the amino acid sequence shown in SEQ NO.2 is derived from SEQ NO.2 by substitution, deletion or addition of one or more amino acids A protein with transaminase activity that has a highly stereoselective-R configuration catalytic activity; wherein, a high degree of stereoselectivity means that the content of one stereoisomer is at least 1.1 times that of the other.

Further, the nucleotide sequence includes one of the following sequences: (1) the nucleotide sequence shown in SEQ NO. 1; (2) having at least 90% similarity with the nucleotide sequence shown in SEQ NO. % identity and encoding a nucleotide sequence of an aminotransferase with highly stereoselective R-configuration catalytic activity; (3) hybridizing to the nucleotide sequence shown in SEQ NO. 1 under high stringency conditions and encoding a highly stereoselective The nucleotide sequence of a transaminase with catalytic activity in the R configuration; wherein, high stereoselectivity means that the content of one stereoisomer is at least 1.1 times that of the other.

The invention provides a recombinant vector in which any of the above nucleotides is effectively connected.

Further, the recombinant vector is pET28b-PamAT.

The present invention provides a host cell transformed or transfected with any of the above recombinant vectors.

The invention provides a method for synthesizing R-aminase derived from ammonia-oxidizing bacterium Psendonocardiaammonioxydans. The method includes the following steps: making ketone compounds, R-aminase or modifications thereof, functional equivalents, and functional Fragment or variant, pyridoxal phosphate and amino donor react in the reaction system, thereby obtaining R-chiral amine.

Further, the above ketone compounds are Wherein R1 and R2 are each independently a C1-C8 alkyl group, a C5-C10 cycloalkyl group, a C5-C10 aryl group or a C5-C10 heteroaryl group, or one of R1 and R2 is a carboxyl group.

Furthermore, the above reaction system also contains a promoter, and the promoter is dimethyl sulfoxide.

Further, the above-mentioned C1-C8 alkyl group is a C1-C8 linear alkyl group, and the amino donor is isopropylamine or D-alanine.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention can efficiently synthesize R-configured chiral amines with high chiral purity by utilizing R-aminase or its modifications, functional equivalents, functional fragments or variants with high stereoselectivity, and is suitable for chiral amines. Industrial production of chiral amines; R-aminase is a rarer type of ω-aminase than S-aminase and has special significance in the biomanufacturing of chiral amines and unnatural amino acids; the new R-aminase HFO announced by the present invention has strict (R) stereoselectivity, and has a broad substrate spectrum, showing industrial application prospects in the green production of chiral amines and non-natural amino acids. The present invention amplifies ammonia-oxidizing Pseudonocardia through PCR technology The gene of the new R-aminase was obtained, and NCBI BlAST base sequence comparison proved that the base sequence of the R-aminase gene indeed belongs to the R-aminase gene sequence of Pseudonocardia ammonia-oxidizing bacteria, and the expression R was obtained through genetic engineering technology. - E.coli DE3-pET-28b-PamAT recombinant strain of transaminase protein can induce expression to obtain R-aminase PamAT. The preparation method is simple and easy to operate. PamAT can tolerate 40% DMSO and still has more than 90% activity. It has Better activity. At the same time, the present invention is based on highly stereoselective PamAT, and uses ketone compounds, amino compounds and pyridoxal phosphate as raw materials to prepare chiral amines or unnatural amino acids. The reaction conditions are mild and conforms to the concept of green chemistry. , providing an important foundation for research, transformation and industrial application, conducive to large-scale industrial promotion, and greatly improving the economic value and social value.

Description of the drawings

Figure 1 is a plasmid map constructed by R-aminase of the present invention.

Figure 2 is the amplification result of the target gene of R-aminase of the present invention.

Figure 3 shows the protein purification results of the recombinant expression of R-aminase of the present invention.

Figure 4 is the characterization of the enzymatic properties of R-aminase of the present invention.

Figure 5 is the HPLC analysis result of the product catalyzed by R-aminase of the present invention.

Figure 6 is the mass spectrum result of the product catalyzed by R-aminase of the present invention.

Figure 7 is a chiral analysis of the product catalyzed by R-aminase of the present invention.

Detailed ways

Parts of the present invention will be further described below in conjunction with examples and reference figures to facilitate those skilled in the art to further understand the present invention, but should not be understood as limiting the present invention. Based on the embodiments of the present invention, all other embodiments obtained without any creative work fall within the protection scope of the present invention.

In order to meet the demand for the preparation of chiral amine compounds, as shown in Figures 1-7, the present invention developed an R-aminase with a high degree of R-configuration stereoselectivity derived from Psendonocardia ammonioxydans. The preparation method of the transaminase is named PamAT. The above-mentioned transaminase is obtained by the present invention using molecular biology technology. Without changing the amino acid sequence, the nucleoside of the transaminase gene derived from Pseudonocardioma ammonia-oxidizing bacteria is The acid sequence was optimized and transformed. The nucleotide sequence of the PamAT gene is shown in SEQ NO. 1, and the amino acid sequence is shown in SEQ NO. 2.

Example 1: Preparation of R-aminase:

(1) Construction of R-aminothermase PamAT vector derived from ammonia-oxidizing Pseudonocardiomas:

The R-aminotransferase PamAT gene sequence of the present invention is derived from the GeneBank database wp-093355841.1. Without changing the amino acid sequence, the codons of the transaminase gene derived from Pseudonocardioma ammonia-oxidizing bacteria are first optimized and restriction endonucleation is used. The enzymes Nco I and Hind III were connected (Sangon Bioengineering (Shanghai) Co., Ltd.) to the pET-28b vector. The plasmid map is shown in Figure 1, the R-aminase nucleic acid sequence is shown in SEQ NO.1, and the amino acid sequence is shown in SEQ As shown in NO.2, transform the pET-28b vector into competent cells of Escherichia coli DH5α strain, resuscitate on a shaking table, spread on an LB culture dish containing kana at a final concentration of 50 μg/ml, and culture in a 37°C incubator overnight. Pick a single colony on the above-mentioned petri dish and inoculate it into LB liquid medium containing kana at a final concentration of 50 μg/ml. Cultivate overnight at 37°C and 220r/min with shaking. Extract the plasmid and identify it by PCR. The upstream primer is AAAATGGGCAGCAGCCATATGACCCTGGCGGAC, and the downstream primer is AAAATGGGCAGCAGCCATATGACCCTGGCGGAC. It is TGCGGCCGCAAGCTTACGATCAACGTC. The identification results are shown in Figure 2. The size of the PCR amplification product is close to the expected 1038bp, indicating that the PamAT vector was successfully constructed.

Figure 2 shows the gel electrophoresis pattern of PCR amplification of the R-aminase target gene, in which Band 1 and Band 2 are the amplified R-aminase target gene, and Band 3 is DNA Marker. Based on the theoretical size of the target fragment being 1108 bp, it can be judged that the size of the amplified product is in line with expectations.

(2) Expression and purification of R-aminase protein PamAT:

The recombinant plasmid pET-28b-PamAT obtained in the above step (1) was transformed into the competent cells of E. coli expression strain BL21 (DE3) using the heat shock method. Inoculate BL21(DE3)-pET28b-PamAT into 100 ml LB medium and culture it at 37°C overnight to obtain a large number of strains. The obtained expression strain was inoculated into LB liquid medium containing kana with a final concentration of 50 μg/ml according to an inoculation amount of 1% and expanded culture at 37°C, 220r/min. When the OD ₆₀₀ value was 0.6, add a final concentration of 0.5mmol. /L IPTG, induce the expression of R-aminase at 28°C, and collect the bacteria by centrifugation after 16 hours. The bacteria are broken by high pressure and Ni affinity chromatography to obtain the R-aminase protein. The size is verified by SDS-PAGE electrophoresis, as shown in the figure. As shown in Figure 3, the molecular weight of the R-aminase protein obtained is about 40kd, which is consistent with the theoretical predicted value of 37.6kd, indicating that we have successfully purified the R-aminase protein. The next step will be to characterize the enzymatic properties of R-aminotransferase PamAT.

Figure 3 shows the SDS-PAGE image of Ni affinity chromatography recombinantly expressing R-aminase. The bands shown in the figure from left to right are the bacterial liquid before induction of R-aminase PamAT (DE3) and the fermentation liquid after induction. , broken supernatant, broken pellet, flow-through, 20mmol/L imidazole protein eluate, 200mmol/L imidazole protein eluate, 400mmol/L imidazole protein eluate and the matrix sample after elution.

(3) Characterization of enzymatic properties of R-aminase PamAT:

Definition of enzyme activity: The amount of enzyme consumed by R-aminase to catalyze the production of 1 μmol of product per minute under certain temperatures and conditions is defined as 1 U.

Preparation of transamination reaction solution: Prepare 50mmol/L phenylethylamine and 50mmol/L pyruvate reaction solution. The final concentration of PLP is 2mmol/L. The pH of the reaction solution is 7.5; the derivatization of the reaction product: the pH of the boric acid derivative solution is 9.0:0.05. Mol/L borax solution and 0.2mol/L boric acid solution are mixed according to the volume ratio of 4:1 to obtain a derivatization buffer solution; phosphate equilibrium buffer pH 7.0: weigh 1.7g potassium dihydrogen phosphate, dissolve it in 300ml ultrapure water, and remove 0.1mol /L sodium hydroxide solution 72.75ml; mix well, add water to make the volume to 500ml; use acetonitrile to make 1% DNFB to the volume; derivatization step: take 5ml of amino acid standard or sample, 5ml of pH9.0 boric acid derivatization buffer, 1% DNFB Take 5 ml of the derivative solution, mix well, seal, protect from light, and react in a 60°C water bath for 60 minutes. After cooling to room temperature, adjust the volume to 50 ml with pH 7.0 phosphate equilibrium buffer, let it stand for 15 minutes, and filter to take the remaining filtrate.

Optimum pH value detection of R-aminase PamAT: Prepare 9 groups of transamination reaction solutions using 50mmol/L phenylethylamine, 50mmol/L pyruvate, 2mmol/LPLP, and deionized water as raw materials, and use KOH to adjust the pH of the reaction solution to 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, and 10, take 490 μl of the transamination reaction solution, add 10 μl of PamAT enzyme solution, and react at 30°C for 5 hours. The reaction mixture is prepared as described above ( The method in 3) was used to measure the production amount of the product D-alanine and calculate the relative activity under different pH conditions. The results are shown in Figure 4. The results show that the optimal pH of R-aminase PamAT is 7.5.

Determination of optimal temperature of R-aminase PamAT: Use 50mmol/L phenylethylamine, 50mmol/L pyruvate, 2mmol/LPLP, and deionized water as raw materials; prepare a pH7.5 transamination reaction solution, and take 490 μl of the transamination reaction solution. Add 10 μl of PamAT enzyme solution and react for 5 hours under different temperature conditions such as 4°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, and 60°C. The reaction mixture was used to measure the amount of product D-alanine produced according to the method in (4) above, and the relative activity under different temperature conditions was calculated. The results are shown in Figure 4. The optimal reaction temperature of the aminotransferase PamAT is 30-35 ℃.

Drawing of R-aminase kinetic curve: fixed donor phenylethylamine content 50mmol/L, 2mmol/L PLP, acceptor pyruvate concentration set to 10mM, 20mM, 40mM, 80mM, 120mM, 160mM, 200mM, 240mM, using KOH Adjust the pH to 7.5, fix the content of acceptor pyruvate at 50mmol/L, 2mmol/L PLP, set the concentration of donor isopropylamine hydrochloride to 10mM, 20mM, 40mM, 80mM, 120mM, 160mM, 200mM, 240mM, and adjust the pH with KOH to 7.5, take 490 μl of each transamination reaction solution, add 10 μl of PamAT enzyme solution, and react for 5 hours at 30°C. Use the reaction mixture to measure the product D-alanine according to the method in (4) above. The amount of production was calculated, the relative activity under different substrate concentration conditions was calculated, and the kinetic curve was automatically fitted using the Michaeli-Menten algorithm in GraphPad.

R-aminase organic solvent tolerance test: Use 50mmol/L phenylethylamine, 50mmol/L pyruvate, 2mmol/LPLP, and deionized water as raw materials to prepare a transamination reaction solution. Add DMSO, methanol, and acetonitrile respectively. Each organic solvent The solvent concentration gradient is 20%, 40%, and KOH adjusts the pH value to 7.5. Take 490 μl of the transamination reaction solution, add 10 μl of PamAT enzyme solution, and react for 5 hours at 30°C. The reaction mixture is prepared as described above ( 4) to measure the production of product D-alanine and calculate the relative activity under the organic solvent content. The results are shown in Figure 4. The aminotransferase PamAT can tolerate 40% DMSO, but methanol and acetonitrile are not effective for PamAT. Enzyme activity is strongly inhibited.

Detection of metal ion tolerance of R-aminase: Use 50mmol/L phenylethylamine, 50mmol/L pyruvate, 2mmol/LPLP, and deionized water as raw materials to prepare a transamination reaction solution, and add Na ⁺ , K ⁺ , and Mg ² respectively. ⁺ , Zn ²⁺ , Mn ²⁺ , Cu ²⁺ , Co ²⁺ , Ni ²⁺ , Fe ³⁺ and other metal ions 10mM, and adjust the pH value to 7.5, take 490μl of the transamination reaction solution, and add 10μl of PamAT enzyme solution, react for 5 hours at 30°C, and use the reaction mixture to measure the amount of product D-alanine produced according to the method in (4) above, and calculate the relative activity of the aminotransferase PamAT under different metal ion conditions. The results are as follows As shown in Figure 4, the metal ions Zn ²⁺ , Cu ^{2 +} , Co ²⁺ , and Ni ²⁺ strongly inhibit enzyme activity, and have good tolerance to Na ⁺ and K ⁺ , and are resistant to Mg ²⁺ , Mn ²⁺ , and Fe ³⁺ enzyme activity is inhibited by about 50%.

(4) Identification of amino donor substrate spectrum of R-aminase PamAT

Reaction simplified formula:

Reaction conditions: 1ml reaction system, add 50mmol/L amino donor, 50mmol/L pyruvic acid, 2mmol/L PLP, and deionized water as raw materials to prepare a transamination reaction solution, adjust the pH to 7.5, and take 490 μl of the transamination reaction solution , add 10 μl of PamAT enzyme solution, and react for 5 hours at 30°C.

Calculation of activity: Use the reaction mixture to measure the amount of product D-alanine produced according to the method (3) above to calculate the relative activity of each substrate.

Example 2: PamAT catalyzes the donor D-serine and the acceptor pyruvate to generate D-alanine and 3-hydroxypyruvate:

Use 50mmol/L D-serine, 50mmol/L pyruvate, 2mmol/L PLP, and deionized water as raw materials to prepare a transamination reaction solution. Adjust the pH to 7.5. Take 490 μl of the transamination reaction solution and add 10 μl of PamAT enzyme solution. , reacted for 5 hours at 30°C, and the reaction mixture was used to measure the amount of product D-alanine produced according to the method (3) above.

Example 3: PamAT catalyzes the donor D-alanine and the acceptor 3-hydroxypyruvate to generate D-serine:

Use 50mmol/L D-alanine, 50mmol/L 3-hydroxypyruvic acid, 2mmol/L PLP, and deionized water as raw materials to prepare a transamination reaction solution. Adjust the pH to 7.5. Take 490 μl of the transamination reaction solution and add 10 μl of The PamAT enzyme solution was reacted for 5 hours at 30°C. The reaction mixture was used to measure the amount of product D-serine produced according to the method in (3) above.

Example 4: PamAT catalyzes the donor isopropylamine and the acceptor pyruvate to generate D-alanine and the by-product acetone:

Use 50mmol/L isopropylamine, 50mmol/L pyruvate, 2mmol/L PLP, and deionized water as raw materials to prepare a transamination reaction solution. Adjust the pH to 7.5. Take 490 μl of the transamination reaction solution, add 10 μl of PamAT enzyme solution, and add React for 5 hours at 30°C, and measure the amount of product D-alanine produced from the reacted mixture according to the method in (3) above.

Example 5: PamAT catalyzes the donor R-phenylethylamine and the acceptor pyruvate to generate acetophenone and isopropylamine:

Use 50mmol/LR-acetophenone, 50mmol/L pyruvate, 2mmol/L PLP, and deionized water as raw materials to prepare a transamination reaction solution. Adjust the pH to 7.5. Take 490 μl of the transamination reaction solution and add 10 μl of PamAT enzyme. Liquid, react for 5 hours at 30°C. Since acetophenone has a strong absorption peak at 245nm, the reaction mixture is detected at a wavelength of 245nm using a microplate reader, and acetone is calculated based on the measured OD ₂₄₅ . Activity of PamAT when acting as a receptor.

Example 6: PamAT catalyzes the donor sec-butylamine and the acceptor pyruvate to generate D-alanine and by-product butanone:

Use 50mmol/L sec-butylamine, 50mmol/L pyruvate, 2mmol/L PLP, and deionized water as raw materials to prepare a transamination reaction solution. Adjust the pH to 7.5. Take 490 μl of the transamination reaction solution and add 10 μl of PamAT enzyme solution. , reacted for 5 hours at 30°C, and measured the amount of product D-alanine produced in the reacted mixture according to the method in (3) above.

Example 7: PamAT catalyzes the donor 2-pentylamine and the acceptor pyruvate to generate D-alanine and the by-product 2-pentanone:

Use 50mmol/L 2-pentylamine, 50mmol/L pyruvate, 2mmol/L PLP, and deionized water as raw materials to prepare a transamination reaction solution. Adjust the pH to 7.5. Take 490 μl of the transamination reaction solution and add 10 μl of PamAT enzyme. liquid, react at 30°C for 5 hours, and measure the amount of product D-alanine produced in the reacted mixture according to the method in (3) above.

(5) Identification of substrate spectrum of R-aminase PamAT amino receptor

Reaction simplified formula:

Reaction conditions: 1ml of reaction system, add 50mmol/L amino acceptor, 50mmol/L R-phenylethylamine, 2mmol/LPLP, and deionized water as raw materials to prepare a transamination reaction solution, adjust the pH to 7.5, and take 490 μl of transamination To the reaction solution, add 10 μl of PamAT enzyme solution and react at 30°C for 5 hours.

Calculation of activity: Use the reaction mixture to measure the amount of product produced according to the following method to calculate the relative activity of each substrate.

Example 8: PamAT catalyzes the donor R-phenylethylamine and the acceptor 2-pentanone to generate 2-pentylamine and the by-product acetophenone:

Use 50mmol/L R-phenylethylamine, 50mmol/L 2-pentanone, 2mmol/L PLP, and deionized water as raw materials to prepare a transamination reaction solution. Adjust the pH to 7.5. Take 490 μl of the transamination reaction solution and add 10 μl The PamAT enzyme solution was reacted for 5 hours at 30°C. Since acetophenone has a strong absorption peak at 245nm, the reaction mixture was detected using a microplate reader at a wavelength of 245nm. According to the measured OD ₂₄₅ To calculate the activity of PamAT when 2-pentanone is used as the receptor.

Example 9: PamAT catalyzes the donor R-phenylethylamine and the acceptor pyruvate to generate D-alanine and the by-product acetophenone:

Use 50mmol/L R-phenylethylamine, 50mmol/L pyruvic acid, 2mmol/L PLP, and deionized water as raw materials to prepare a transamination reaction solution. Adjust the pH to 7.5. Take 490 μl of the transamination reaction solution and add 10 μl of PamAT. The enzyme solution was reacted for 5 hours at 30°C. The reaction mixture was used to measure the amount of product D-alanine produced according to the method in (3) above.

Example 10: PamAT catalyzes the donor R-phenylethylamine and the acceptor 3-hydroxypyruvate to generate D-alanine and the by-product acetophenone:

Use 50mmol/L R-phenylethylamine, 50mmol/L 3-hydroxypyruvic acid, 2mmol/L PLP, and deionized water as raw materials to prepare a transamination reaction solution. Adjust the pH to 7.5. Take 490 μl of the transamination reaction solution and add 10 μl. The PamAT enzyme solution was reacted for 5 hours at 30°C. The reaction mixture was used to measure the amount of product D-alanine produced according to the method in (3) above.

In order to confirm that the catalyzed product of R-aminase is D-alanine, the reaction mixture was subjected to HPLC, mass spectrometry and chiral chromatography. The detection of the theoretical product alanine requires pre-column derivatization and then HPLC analysis. Derivatives of the alanine standard are also prepared as positive controls. The results are shown in Figure 5. (a) The arrow in the figure points to the peak of the alanine standard derivative. (b) The arrow in the figure points to the peak of the derivatized reaction product. The retention time is the same as that of the alanine standard derivative. The retention times of the amino acid standard derivatives are consistent, and it is speculated that the reaction product of PamAT may contain alanine.

However, in order to directly determine that the generated product is alanine, its relative molecular mass needs to be accurately measured. Therefore, it is necessary to purify the derivatized product and send it to a third party for mass spectrometry detection. The results are shown in Figure 6. The molecular mass of alanine after derivatization is 255.1, which is consistent with the molecular mass of 254.1 detected in the figure. In addition, mass spectrometry analysis determined that the compound with a molecular mass of 509.2 is actually a dimer of derivatized alanine. Therefore, it can be determined that the aminotransferase PamAT can catalyze the generation of alanine, but the mass spectrometry cannot analyze the chirality.

In order to determine whether the generated product alanine is D-form or L-form, it needs to be analyzed by chiral column chromatography. The product generated by the transamination reaction was derivatized and purified. At the same time, the L-alanine standard and D-alanine standard were derivatized and purified. The three processed samples were handed over to a third party for chiral column detection. . The detection results are shown in Figure 7. The arrow in (a) points to the peak of the L-alanine standard derivative, and its corresponding retention time is 9.677 min; the arrow in (b) points to the peak of D-alanine. The peak of the amino acid standard derivative has a corresponding retention time of 13.016 min; the two arrows in the figure (c) point to the peaks of the reaction products after derivatization, and the peak pointed to by the left arrow has a corresponding retention time of 9.723 min. , indicating that the component is an L-alanine derivative. The corresponding retention time of the peak pointed by the arrow on the right is 13.092 min, indicating that the component is a D-alanine derivative. After calculation, D-alanine in the reaction product The ee value is 78%. The aminotransferase PamAT can catalyze the production of D-alanine using phenylethylamine as the amino donor and pyruvate as the amino acceptor. Therefore, the aminotransferase PamAT is an R-selective aminotransferase (R-ATA).

The present invention can use D-amino acid and R-amine as amino donors and adopt an asymmetric synthesis method of adding ammonia to chiral carbonyl compounds to generate chiral amines and non-natural amino acids, for which the R-aminase PamAT has catalytic activity. Amino acids or R-amines: Among them, those with better catalytic activity include D-alanine, D-serine, isopropylamine, R-phenylethylamine, 2-aminopentane, 3-aminopentylamine, pyruvic acid, 3- Hydroxypyruvic acid, acetone, etc.

From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the novel transaminase disclosed in the present invention catalyzes the transfer of amino groups in the amino donor to prochiral ketones or aldehydes, thereby producing the corresponding R Using the novel aminotransferase of the present invention to prepare chiral amines can not only convert more substrates, but also obtain R-configured chiral amines with high purity and stability above 98%. The raw materials used in the synthesis method are easily available, the method is simple, the chemical reaction conditions are mild, the yield and the purity of the enantiomers are very high, the entire production process is simple to operate, and it is a feasible and less polluting synthesis process. The preparation of chiral amines provides a new approach and method.

For specific PamAT activity determination, please refer to the following Table 1-Table 2:

Table 1 Determination of activity of aminotransferase PamAT towards amino donors

Table 2 Determination of activity of aminotransferase PamAT on receptor ketones

The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention. should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

sequence list

<110> Jiangsu Ocean University

<120> An R-aminase derived from ammonia-oxidizing Pseudonocardiomonas and its synthesis method

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1038

<212> DNA

<213> Psendonocardia ammonioxydans

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gacacctcca gcccctacgc cggaggcgcg gcgtggatcg aaggcgagta cgtcccggcg 180

tcggaggccc ggatctccat cttcgacacc ggcttcggcc actccgacct gacctacacc 240

gtcgcccacg tctggcacgg caacatcttc cggctggccg accacatcga acgcctcctc 300

gacggagccc ggaagctgcg gctcgcctcg ccgtacgacg agaccgagat cgccgagatc 360

gcgaaacgct gtgtcggtct gtcccaattg cgcgaggcct atgtgaacat cacgctcacc 420

cgcggctacg gcaagcggaa gggcgagaag gatctgagca agctcacctc gcagatctac 480

gtctacgcga tcccgtacct gtgggcgttc cctccgtacg aacagatctt cgggacctcc 540

gcggtcgtac cccgccacgt gcaacgcgcc gggcgcaaca ccatcgatcc gacgatcaag 600

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gtggtcaagg acggcgcgct ggcgtccccg tcccggaacg cgctacccgg gatcacccgc 780

aagaccgtct tcgagatcgc ccacgcgcga gggatctcgg ccgagttgcg cgacgtcacg 840

agccgggagc tctacgacgc cgacgagttg atggccgtca cgacggcggg cggagtcacc 900

ccgatcacct cgctcgacgg cgccgctgtc ggcgacggcg agccgggccc gatcacggtg 960

gcgatccggg accggttctg ggcgctcatg gacgagccgt cggacttgat cgacacgatc 1020

aggtacgacg tggatcgc 1038

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<213> Psendonocardia ammonioxydans

<400> 2

Met Thr Leu Ala Asp Ser Gly Thr Asp Phe Ser Thr Ser Asn Leu Val

1 5 10 15

Ala Val Glu Pro Gly Ala Ile Arg Glu Asp Thr Pro Pro Gly Ser Val

20 25 30

Ile Gln Tyr Ser Asp Tyr Glu Leu Asp Thr Ser Ser Pro Tyr Ala Gly

35 40 45

Gly Ala Ala Trp Ile Glu Gly Glu Tyr Val Pro Ala Ser Glu Ala Arg

50 55 60

Ile Ser Ile Phe Asp Thr Gly Phe Gly His Ser Asp Leu Thr Tyr Thr

65 70 75 80

Val Ala His Val Trp His Gly Asn Ile Phe Arg Leu Ala Asp His Ile

85 90 95

Glu Arg Leu Leu Asp Gly Ala Arg Lys Leu Arg Leu Ala Ser Pro Tyr

100 105 110

Asp Glu Thr Glu Ile Ala Glu Ile Ala Lys Arg Cys Val Gly Leu Ser

115 120 125

Gln Leu Arg Glu Ala Tyr Val Asn Ile Thr Leu Thr Arg Gly Tyr Gly

130 135 140

Lys Arg Lys Gly Glu Lys Asp Leu Ser Lys Leu Thr Ser Gln Ile Tyr

145 150 155 160

Val Tyr Ala Ile Pro Tyr Leu Trp Ala Phe Pro Pro Tyr Glu Gln Ile

165 170 175

Phe Gly Thr Ser Ala Val Val Pro Arg His Val Gln Arg Ala Gly Arg

180 185 190

Asn Thr Ile Asp Pro Thr Ile Lys Asn Tyr Gln Trp Gly Asp Leu Thr

195 200 205

Ala Ala Ser Phe Glu Ala Lys Asp Arg Gly Ala Arg Thr Gly Ile Leu

210 215 220

Leu Asp Ala Asp Gly Cys Val Ala Glu Gly Pro Gly Phe Asn Val Val

225 230 235 240

Val Val Lys Asp Gly Ala Leu Ala Ser Pro Ser Arg Asn Ala Leu Pro

245 250 255

Gly Ile Thr Arg Lys Thr Val Phe Glu Ile Ala His Ala Arg Gly Ile

260 265 270

Ser Ala Glu Leu Arg Asp Val Thr Ser Arg Glu Leu Tyr Asp Ala Asp

275 280 285

Glu Leu Met Ala Val Thr Thr Ala Gly Gly Val Thr Pro Ile Thr Ser

290 295 300

Leu Asp Gly Ala Ala Val Gly Asp Gly Glu Pro Gly Pro Ile Thr Val

305 310 315 320

Ala Ile Arg Asp Arg Phe Trp Ala Leu Met Asp Glu Pro Ser Asp Leu

325 330 335

Ile Asp Thr Ile Arg Tyr Asp Val Asp Arg

340 345

Claims (6)

1. A method for synthesizing R-type chiral amine by using R-aminotransferase from pseudomonas ammoxidation, which is characterized in that: the method comprises the following steps: the pyruvic acid, 3-hydroxy pyruvic acid, acetone, ketone compound of 2-pentanone, R-aminotransferase, pyridoxal phosphate and amino donor are reacted in a reaction system, thereby obtaining R-type chiral amine, the enzyme is named PamAT, and the amino acid sequence of the R-aminotransferase is shown as SEQ ID NO. 2.

2. A process for the synthesis of chiral R-amines using an R-aminotransferase from pseudomonas ammoxidation according to claim 1, wherein: the nucleotide sequence of the coded PamAT is shown as SEQ ID NO. 1.

3. A process for the synthesis of chiral R-amines using an R-aminotransferase from pseudomonas ammoxidation according to claim 2, wherein: the recombinant vector is effectively connected with a nucleotide sequence shown as SEQ ID NO. 1.

4. A process for the synthesis of chiral R-amines using an R-aminotransferase from pseudomonas ammoxidation according to claim 3, wherein: the recombinant vector is pET28b-PamAT.

5. The method for synthesizing R-type chiral amine by using R-aminotransferase from Pseudomonas ammoxidation according to claim 4, wherein: the recombinant vector transfects or transforms a host cell.

6. A process for the synthesis of chiral R-amines using an R-aminotransferase from pseudomonas ammoxidation according to claim 1, wherein: the reaction system also contains a cosolvent, and the cosolvent is dimethyl sulfoxide.