R-type transaminase mutant and application

Abstract

This invention discloses an R-type transaminase mutant and its applications, relating to the field of biocatalysis technology. The amino acid sequence of the R-type transaminase of this invention is shown in SEQ ID NO.7, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1, and the amino acid sequences of the R-type transaminase mutant are shown in SEQ ID NO.2-SEQ ID NO.6. This invention also provides the application of the R-type transaminase mutant in the asymmetric synthesis of chiral amine compounds. The R-type transaminase of this invention has a maximum sequence similarity of only about 80% with existing R-type transaminases. It provides a method for the enzymatic synthesis of chiral intermediates, with simple reaction steps, mild reaction conditions, and a single-step reaction to reduce and amination 1-naphthyl acetone to a high optical purity (…). R )-1-(1-naphthyl)-ethylamine, with a conversion rate of over 90% and an ee value (enantiomer excess) greater than 99%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological catalysis, and in particular to an R-type transaminase mutant and application. BACKGROUND

[0002] Chiral amines are intermediates of many important medicines and fine chemicals, and play an important role in today's drug research and development process. Chiral amines are often used to synthesize chiral drugs, such as rivastigmine for treating Alzheimer's disease, sitagliptin as the main component of an antidiabetic drug, and cinacalcet for treating hypercalcemia. The application of these chiral compounds in drug synthesis greatly promotes the exploration of efficient synthesis of chiral amine drugs.

[0003] R (R)-1-(1-naphthyl)-ethylamine is an important chiral aromatic amine and a core building block of pharmaceutical intermediates. For example, (R)-1-(1-naphthyl)-ethylamine and 3-(3-trifluoromethylphenyl)-propionic acid can be coupled to produce the calcimimetic agent cinacalcet, which can treat secondary hyperparathyroidism and hypercalcemia associated with parathyroid carcinoma. At present, the synthesis of (R)-1-(1-naphthyl)-ethylamine still mainly relies on traditional chemical synthesis, which mainly uses (D)-tartaric acid as a resolving agent to separate 1-(1-naphthyl)-ethylamine, but this process is complicated, the optical purity of the product is low, multiple recrystallizations are required, and a large amount of waste is generated. R R Compared with existing chemical synthesis processes, biological catalysis can well make up for the shortcomings of chemical methods, and has the advantages of simple synthesis steps, no need for harsh conditions, green and pollution-free, high stereoselectivity, etc. As an important biological catalyst, ω-transaminase can asymmetrically synthesize optically pure chiral amines from ketone or aldehyde substrates through a simple one-step reaction, and has the advantages of mild reaction conditions, high stereoselectivity, and wide substrate spectrum, making it an important enzyme resource for the synthesis of chiral amines. For example, Chinese Patent No. CN111411096A discloses a transaminase catalyst and a method for enzymatic synthesis of (R)-1-naphthylamine, which discovers a new transaminase catalyst that can directly produce (R)-1-(1-naphthyl)ethylamine under mild conditions without chemical resolution; Chinese Patent No. CN119614531A discloses the application of R-transaminase and its mutants in the asymmetric synthesis of chiral amine compounds, which obtains excellent mutants with improved substrate loading and space-time yield by using point mutation technology to modify wild-type ω-transaminase.

[0004] Compared with existing chemical synthesis processes, biological catalysis can well make up for the shortcomings of chemical methods, and has the advantages of simple synthesis steps, no need for harsh conditions, green and pollution-free, high stereoselectivity, etc. As an important biological catalyst, ω-transaminase can asymmetrically synthesize optically pure chiral amines from ketone or aldehyde substrates through a simple one-step reaction, and has the advantages of mild reaction conditions, high stereoselectivity, and wide substrate spectrum, making it an important enzyme resource for the synthesis of chiral amines. For example, Chinese Patent No. CN111411096A discloses a transaminase catalyst and a method for enzymatic synthesis of (R)-1-naphthylamine, which discovers a new transaminase catalyst that can directly produce (R)-1-(1-naphthyl)ethylamine under mild conditions without chemical resolution; Chinese Patent No. CN119614531A discloses the application of R-transaminase and its mutants in the asymmetric synthesis of chiral amine compounds, which obtains excellent mutants with improved substrate loading and space-time yield by using point mutation technology to modify wild-type ω-transaminase. R

[0005] ​​​To address the shortcomings of existing technologies, transaminases have shown great potential in the synthesis of chiral amines. Therefore, there is an urgent need to obtain more ω-transaminases to enable efficient and highly stereoselective catalytic synthesis of cinacalcet drug intermediates under extreme conditions. R )-1-(1-naphthyl)-ethylamine, to meet the needs of industrial production. Summary of the Invention

[0006] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a solution. R Type I transaminase mutants and their applications, specifically providing a type I transaminase resistant to strong alkalis. R Type I transaminase mutants and their application in the synthesis of chiral amine drug intermediates for cinacalcet, compared with existing catalytic synthesis ( R )-1-(1-naphthyl)-ethylamine R The maximum sequence similarity of the type transaminase is only about 80%. This study also provides a method for the bioenzymatic synthesis of chiral intermediates, which involves simple reaction steps, mild reaction conditions, and a single-step reaction to reduce and amination 1-naphthylacetone to high optical purity. R )-1-(1-naphthyl)-ethylamine, with a conversion rate of over 90% and an ee value (enantiomer excess) greater than 99%.

[0007] Specifically, this invention relates to a source Gordonia sp .of R Type I transaminases, mutants, their preparation methods, and recombinants R Type II transaminase mutant catalyst, and the catalyst utilizing this R Type II transaminase and mutant catalyst, using 1-naphthyl ethylone as substrate and isopropylamine as amino donor, asymmetric catalytic generation ( R A method for synthesizing 1-(1-naphthyl)-ethylamine. This biocatalyst exhibits excellent catalytic performance at high concentrations of isopropylamine. Significant improvements in catalytic efficiency can be achieved by mutating only one or two amino acid sites. Furthermore, thanks to its good thermal stability and strong alkali resistance, this enzyme shows promising industrial application prospects in the synthesis of chiral amine drug intermediates, cinacalcet.

[0008] The technical solution of the present invention is as follows:

[0009] The first aspect of the present invention provides R The amino acid sequences of the type transaminase mutant are shown in SEQ ID NO.2-SEQ ID NO.6.

[0010] R The amino acid sequence of type 1 transaminase mutants is obtained through R It is obtained by mutating one or more amino acid residues at positions 73 and 149 of the amino acid sequence of type transaminase.

[0011] Preferably, the R The type 1 transaminase mutant has an amino acid sequence as shown in SEQ ID NO. 7. R It is obtained by mutation of type transaminase.

[0012] Preferably, the encoding of R The nucleotide sequence of the gene for type I transaminase is shown in SEQ ID NO. 1.

[0013] The second aspect of the present invention provides R A method for preparing type I transaminase mutants includes the following steps:

[0014] S1, with the amino acid sequence shown in SEQ ID NO.7 R Using a plasmid of type I transaminase as a template, DNA polymerase was used to polymerize the... R The expression plasmid of type I transaminase was amplified into a whole plasmid to prepare a recombinant expression vector;

[0015] S2. Transform the recombinant expression vector into host cells to prepare the recombinant expression transformant;

[0016] S3. Cultivate the recombinant expression transformant and isolate the expressed... R Type II transaminase mutant.

[0017] Preferably, in step S1, the sequences of the primers used for amplification are shown in SEQ ID NO.8-SEQ ID NO.13.

[0018] Preferably, in step S2, the present invention uses *Escherichia coli* as the host cell, and *Escherichia coli* is the preferred host cell. E E. coli BL21(DE3).

[0019] Preferably, step S3 includes any one of the following specific steps:

[0020] (1) Cultivate the recombinant expression transformant and isolate the contents of the transformant. R Transformed somatic cells of type I transaminase mutant;

[0021] (2) Cultivate the recombinant expression transformant and isolate the contents of the transformant. R Crude enzyme solution of type I transaminase mutant;

[0022] (3) Cultivate the recombinant expression transformant and isolate the contents of the transformant. R Transformed somatic cells of type I transaminase mutant were dried to obtain R Freeze-dried bacterial powder of type transaminase mutant, or purified product R Pure enzyme of type I transaminase mutant.

[0023] A third aspect of the present invention provides a recombinant R Type II transaminase mutant catalyst, the recombinant R The catalyst for the type 1 transaminase mutant includes any one of the following forms:

[0024] (1) Contains the above R Transformed somatic cells of type I transaminase mutant;

[0025] (2) Contains the above R Crude enzyme solution of type I transaminase mutant;

[0026] (3) Contains the above R Freeze-dried bacterial powder of type transaminase mutant;

[0027] (4) Contains the above R Pure enzyme of type I transaminase mutant.

[0028] The fourth aspect of the present invention provides the aforementioned R Type I transaminase mutants or the recombinants R Application of type-3 transaminase mutant catalysts in the asymmetric synthesis of chiral amine compounds.

[0029] Using 1-naphthyl ethylone as a substrate and isopropylamine as an amino donor, the aforementioned enzyme was used as a biocatalyst to asymmetrically catalyze the synthesis of (R)-1-(1-naphthyl)-ethylamine. This biocatalyst exhibits excellent catalytic performance at high concentrations of isopropylamine; a significant increase in catalytic efficiency can be achieved by mutating only one or two amino acid sites. Furthermore, thanks to its good thermal stability and strong alkali resistance, this enzyme shows promising industrial application prospects in the synthesis of chiral amine drug intermediates, cinacalcet.

[0030] A fifth aspect of the present invention provides a method for preparing a chiral amine compound, comprising the following steps:

[0031] Using 1-naphthyl ethyl ketone and isopropylamine as raw materials, the following were added: R Type I transaminase mutants or the recombinants R The type 1 transaminase mutant catalyst is used as a catalyst in the reaction to produce ( R )-1-(1-naphthyl)-ethylamine.

[0032] Preferably, the preparation method includes the following specific steps:

[0033] The R Type I transaminase or the aforementioned R Type I transaminase mutants or the recombinants R The type 1 transaminase mutant catalyst was mixed with 1-naphthyl ethyl ketone, acidified isopropylamine, buffer solution and coenzyme PLP to obtain the reaction solution;

[0034] The reaction solution was placed at 20℃~40℃ and 100 rpm~300 rpm to react, yielding ( R )-1-(1-naphthyl)-ethylamine.

[0035] This invention has at least one of the following beneficial effects:

[0036] This invention provides a transaminase mutant with high catalytic activity and its application. R Type I transaminase mutants can use 1-naphthyl ethylone as a substrate and inexpensive isopropylamine as an amino donor to asymmetricly synthesize cinacalcet chiral amine drug intermediates. R )-1-(1-naphthyl)-ethylamine, the reaction steps are simple, the reaction conditions are mild, and only one step is needed to reduce and amination 1-naphthylacetone to high optical purity ( R The transaminase developed in this invention exhibits a conversion rate exceeding 90% and an enantiomeric excess value (ee value) greater than 99%, achieving a chiral control difficulty, requiring expensive chiral auxiliaries, and leaving heavy metal residues in chemical synthesis methods. Furthermore, the mutant transaminase possesses excellent thermal stability and strong alkali resistance, along with advantages such as high catalytic efficiency, high stereoselectivity, and the absence of other byproducts, making it well-suited for industrial applications and possessing broad application value. Attached Figure Description

[0037] Figure 1 In Example 4 R A schematic diagram illustrating the principle of asymmetric synthesis of cinacalcet intermediates using type 7 transaminase (RATA-7) and its mutants as catalysts.

[0038] Figure 2 In Example 4 R The conversion results of the asymmetric synthesis of cinacalcet intermediates using type 7 transaminase (RATA-7) and its mutants as catalysts.

[0039] Figure 3 In Example 4 R Chromatograms of the asymmetric synthesis of cinacalcet intermediates using type 7 transaminase (RATA-7) and its mutants as catalysts.

[0040] Figure 4 In Example 4 R Characterization diagrams of the optimal temperature, temperature stability, optimal pH, and pH stability of type 7 transaminase (RATA-7). Detailed Implementation

[0041] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0042] Materials and reagents used in genetic engineering: Escherichia coli E. coli BL21(DE3) and plasmid pET-28a(+) were obtained from our laboratory's collection. Plasmid extraction kits and DNA purification and recovery kits were purchased from TOROIVD Technology Co., Ltd.; SDS-PAGE gel rapid preparation kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.; DNA markers were purchased from Beijing TransGen Biotech Co., Ltd.; please refer to the product instructions for use of the above reagents.

[0043] Primer synthesis and sequence sequencing were performed by Zhejiang Youkang Biotechnology Co., Ltd.

[0044] Isopropylamine was purchased from Shanghai Titan Technology, and the rest were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0045] Example 1: Strain Construction

[0046] In this invention R Type I transaminase is transaminase ATA117 ( Arthrobacter sp. Using a template, the gene was extracted from the NCBI database through gene mining and homologous sequence alignment. The entire gene was synthesized, and the Nde I and Hind III restriction sites were selected and inserted into the pET-28a(+) expression vector. The recombinant expression vector containing the target gene was then transformed into *E. coli* using the calcium chloride method. E. coli After BL21(DE3) competent cells are obtained, recombinant cells can be induced to express [the desired expression]. R The genetically engineered bacterium that produces type 7 transaminase, designated RATA-7, is derived from... Gordonia sp. Its sequence similarity to the template transaminase ATA117 is 48%.

[0047] R The nucleotide sequence of type 7 transaminase (RATA-7) is shown in SEQ ID NO.1.

[0048] SEQ ID NO.1:

[0049]

[0050] R The amino acid sequence of type 7 transaminase (RATA-7) is shown in SEQ ID NO.7.

[0051] SEQ ID NO.7:

[0052] MTTSNDIDYSASNLVSVAPGAIREPTPSGSVIQYSEYELDESSPFAGGAAWVEGEYVPAAEARISLFDTGFGHSDLTYTVAHVWHGNIFRLKDHIDRVFDGARKLRLQSPLSKAEVEDITKRCVSLSQLRESFVNITITRGYGARKGEKDLSKLTSQIYIYAIPYLWAFPP EEQIFGTSAIVPRHVRRAGRNTVDPTVKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVVMVKDGKLSSPSRNALPGITRLTVMELAADMGIEFTLRDITSRELYEADELIAVTTAGGITPITSLDGVPLGDGEPGPMTVAIRDRFWALMDEPSPLVEAIEY.

[0053] Example 2 Mutant Construction

[0054] This embodiment includes the content from Embodiment 1. R Using the pET-28a(+) plasmid of type 7 transaminase (RATA-7) as a template, the expression plasmid of RATA-7 was amplified using PrimeSTAR Max high-fidelity DNA polymerase (TAKARA, catalog number R045A). Primers are shown in Table 1. The PCR system consisted of: 1 μL upstream primer (20 μM), 1 μL downstream primer (20 μM), 1 μL template DNA (50 μg / mL), 9.5 μL ddH2O, and 12.5 μL PrimeSTAR Max premix (2×). The PCR experimental procedure is shown in Table 2 below.

[0055] Table 1 Primers used in the construction of RATA-7 mutants

[0056]

[0057] Table 2 Amplification Procedure

[0058]

[0059] The above method was used to obtain a recombinant expression vector containing the gene with the mutation site.

[0060] R The amino acid sequence of the type 7 transaminase (RATA-7) mutant is as described in Example 1. R The RATA-7 mutant is obtained by mutating one or two amino acid residues at positions 73 and 149 of the amino acid sequence. The amino acid sequences of the RATA-7 mutant are shown in SEQ ID NO.2 (H73F), SEQ ID NO.3 (K149L), SEQ ID NO.4 (K149I), SEQ ID NO.5 (H73F / K149L), and SEQ ID NO.6 (H73F / K149I), respectively. The underlined parts in the sequences below are the mutation sites.

[0061] SEQ ID NO.2:

[0062] MTTSNDIDYSASNLVSVAPGAIREPTPSGSVIQYSEYELDESSPFAGGAAWVEGEYVPAAEARISLFDTGFG F SDLTYTVAHVWHGNIFRLKDHIDRVFDGARKLRLQSPLSKAEVEDITKRCVSLSQLRESFVNITITRGYGARKGEKDLSKLTSQIYIYAIPYLWAFPPEEQIFGTSAIVPRHVRRAGRNTVDPTVKNYQWGDLT AASFEAKDRGARTAILLDSDNCVAEGPGFNVVMVKDGKLSSPSRNALPGITRLTVMELAADMGIEFTLRDITSRELYEADELIAVTTAGGITPITSLDGVPLGDGEPGPMTVAIRDRFWALMDEPSPLVEAIEY.

[0063] SEQ ID NO.3:

[0064] MTTSNDIDYSASNLVSVAPGAIREPTPSGSVIQYSEYELDESSPFAGGAAWVEGEYVPAAEARISLFDTGFGHSDLTYTVAHVWHGNIFRLKDHIDRVFDGARKLRLQSPLSKAEVEDITKRCVSLSQLRESFVNITITRGYGARKGE LDLSKLTSQIYIYAIPYLWAFPPEEQIFGTSAIVPRHVRRAGRNTVDPTVKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVVMVKDGKLSSPSRNALPGITRLTVMELAADMGIEFTLRDITSRELYEADELIAVTTAGGITPITSLDGVPLGDGEPGPMTVAIRDRFWALMDEPSPLVEAIEY。

[0065] SEQ ID NO.4:

[0066] MTTSNDIDYSASNLVSVAPGAIREPTPSGSVIQYSEYELDESSPFAGGAAWVEGEYVPAAEARISLFDTGFGHSDLTYTVAHVWHGNIFRLKDHIDRVFDGARKLRLQSPLSKAEVEDITKRCVSLSQLRESFVNITITRGYGARKGE I DLSKLTSQIYIYAIPYLWAFPPEEQIFGTSAIVPRHVRRAGRNTVDPTVKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVVMVKDGKLSSPSRNALPGITRLTVMELAADMGIEFTLRDITSRELYEADELIAVTTAGGITPITSLDGVPLGDGEPGPMTVAIRDRFWALMDEPSPLVEAIEY。

[0067] SEQ ID NO.5:

[0068] MTTSNDIDYSASNLVSVAPGAIREPTPSGSVIQYSEYELDESSPFAGGAAWVEGEYVPAAEARISLFDTGFG F SDLTYTVAHVWHGNIFRLKDHIDRVFDGARKLRLQSPLSKAEVEDITKRCVSLSQLRESFVNITITRGYGARKGE LDLSKLTSQIYIYAIPYLWAFPPEEQIFGTSAIVPRHVRRAGRNTVDPTVKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVVMVKDGKLSSPSRNALPGITRLTVMELAADMGIEFTLRDITSRELYEADELIAVTTAGGITPITSLDGVPLGDGEPGPMTVAIRDRFWALMDEPSPLVEAIEY.

[0069] SEQ ID NO.6:

[0070] MTTSNDIDYSASNLVSVAPGAIREPTPSGSVIQYSEYELDESSPFAGGAAWVEGEYVPAAEARISLFDTGFG F SDLTYTVAHVWHGNIFRLKDHIDRVFDGARKLRLQSPLSKAEVEDITKRCVSLSQLRESFVNITITRGYGARKGE I DLSKLTSQIYIYAIPYLWAFPPEEQIFGTSAIVPRHVRRAGRNTVDPTVKNYQWGDLTAASFEAKDRGARTAILLDSDNCVAEGPGFNVVMVKDGKLSSPSRNALPGITRLTVMELAADMGIEFTLRDITSRELYEADELIAVTTAGGITPITSLDGVPLGDGEPGPMTVAIRDRFWALMDEPSPLVEAIEY.

[0071] Example 3: Strain Culture

[0072] The PCR product of the recombinant vector containing the mutant gene obtained in Example 2 was added to a centrifuge tube containing competent cells, gently mixed by pipetting, and incubated on ice for 30 min. Then, it was heat-shocked at 42°C for 90 s and incubated on ice for 5 min. Then, 900 µL of LB liquid medium was added and cultured at 37°C and 200 rpm for 45 min. After the culture was completed, the culture was centrifuged at 8000 rpm for 2 min, and then 900 µL of supernatant was discarded. The cells were resuspended, spread, and incubated upside down in a 37°C incubator for 12 h to finally obtain positive clones, i.e., recombinant expression transformants.

[0073] The recombinant transformants were inoculated into 5 mL LB liquid medium containing 50 µL / mL kanamycin resistance and incubated overnight at 37°C with a shaker. Then, 500 µL of the overnight culture was added to a 50 mL shake flask containing 50 mL LB liquid medium (containing 50 µL / mL kanamycin resistance) and incubated at 37°C with a shaker for approximately 2 hours. OD 600 The concentration reached approximately 0.6. 0.1 mM IPTG solution was added for induction, followed by 22 h of induction in a shaker at 20°C. After induction, the bacterial culture was transferred to a 50 mL centrifuge tube, balanced, and centrifuged at 8000 rpm for 10 min at 4°C. After centrifugation, the supernatant was discarded, and 30 mL of physiological saline was added to resuspend the cells. The balance was then maintained, and the cells were centrifuged at 8000 rpm for 10 min at 4°C. After centrifugation, the supernatant was discarded, and the collected cells were stored at -80°C for later use. Subsequently, the harvested recombinant cells were added to 8 mL of Tris-HCl buffer (20 mM, pH 8.5, containing 0.1 mM PLP) for sonication, and the supernatant was collected by centrifugation to obtain the crude enzyme solution. This solution was then freeze-dried using a vacuum freeze dryer to obtain the final product. R Freeze-dried bacterial powder of the type 7 transaminase (RATA-7) mutant. Alternatively, the crude enzyme solution can be purified to obtain... R Pure enzyme solution of the RATA-7 mutant transaminase. (Used) R Both the lyophilized bacterial powder and the pure enzyme solution of the type 7 transaminase (RATA-7) mutant can be used to catalyze the asymmetric synthesis of cinacalcet chiral amine drug intermediates.

[0074] Example 4: Synthesis of cinacalcet drug intermediate by asymmetric reductive amination of RATA-7 mutant

[0075] R A schematic diagram illustrating the principle of asymmetric synthesis of cinacalcet intermediates using RATA-7 transaminase and its mutants as catalysts is shown below. Figure 1 As shown, the specific synthesis method includes the following steps:

[0076] 1. Add 10 mg to a 1 mL reaction system R Type I transaminase wild-type (in Example 1) RThe reaction mixture consisted of lyophilized bacterial powder of the mutant prepared in Example 3 (type 3 transaminase), 10 mM 1-naphthyl ethyl ketone, 1 M acidified isopropylamine, Tris-HCl buffer (0.1 M pH 8.0), and 0.1 mM PLP. The reaction solution was placed at 30°C and 200 rpm. At different reaction time points (2h, 5h, 8h, 12h, 24h), 100 μL of the reaction solution was dissolved in 200 μL of a mixed solution of methanol:1M HCl = 3:1 (volume ratio) for quenching, vortexed, centrifuged and filtered, and 200 μL of the supernatant was taken for HPLC analysis.

[0077] Conversion rates at different times, such as Figure 2 As shown, Figure 2 WT in R The results of type I transaminase wild-type, Figure 2 H73F, K149L, K149I, H73F / K149L, and H73F / K149I are respectively those shown in SEQ ID NO.2-SEQ ID NO.6. R The result of type I transaminase mutants, by Figure 2 It can be seen that, R The conversion rates of both wild-type and mutant transaminases increased over time, reaching a maximum at 8 hours. R The wild-type conversion rate of the transaminase was 45%, as shown in SEQ ID NO.3-SEQ ID NO.6. R The conversion rates of all type I transaminase mutants reached over 70%. After 24 hours of reaction, R The wild-type conversion rate of the type transaminase is greater than 80%, as shown in SEQ ID NO.3-SEQ ID NO.6. R The conversion rate of type I transaminase mutants can be as high as 90% or more.

[0078] 2. Add 2 mg / mL of [agent name] to a 1 mL reaction system. R Type I transaminase wild-type (in Example 1) R The reaction mixture consisted of 200 μL of the mutant pure enzyme solution prepared in Example 3, 20 mM 1-naphthyl ethyl ketone, 1 M acidified isopropylamine, Tris-HCl buffer (0.1 M pH 8.0), and 0.1 mM PLP. The reaction solution was incubated at 30°C and 200 rpm for 24 h. After the reaction, 500 μL of the reaction solution was placed in a 70°C water bath for 30 min to terminate the reaction and remove excess isopropylamine. 200 μL of 6 M NaOH solution was added, and the mixture was shaken to mix. Then, an equal volume of ethyl acetate was added for extraction, and the mixture was shaken to mix. The mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was transferred to a new centrifuge tube. The dried product was the target product.R )-1-(1-naphthyl)-ethylamine.

[0079] HPLC chiral analysis showed that their ee values ​​were all greater than 99.9%, and the chromatograms are as follows. Figure 3 As shown, where, Figure 3 The Rac-Amine in the results is a racemic amine standard. Figure 3 RATA-7 in the text is R The results of type I transaminase wild-type, Figure 3 H73F, K149L, K149I, H73F / K149L, and H73F / K149I are respectively those shown in SEQ ID NO.2-SEQ ID NO.6. R Results of type I transaminase mutants.

[0080] 3. Characterization of the enzymatic properties of RATA-7:

[0081] To evaluate the potential of this transaminase for industrial applications, this embodiment also characterizes the enzymatic properties of RATA-7, including optimal temperature, temperature stability, optimal pH, and pH stability, as shown below. Figure 4 A in Figure 4 B in Figure 4 C in Figure 4 As shown in D, the characterization method is as follows:

[0082] The optimal pH of RATA-7 was determined at 30°C within a pH range of 4–11 (using citrate, phosphate, Tris-HCl, and glycine-sodium hydroxide buffers, respectively). The pH stability of the enzyme was determined by incubating it at 30°C in buffers ranging from pH 4 to pH 11 for 30 min and detecting residual enzyme activity.

[0083] The optimal temperature for this enzyme was determined by measuring its activity at different temperatures (4℃, 16℃, 25℃, 30℃, 35℃, 40℃, 50℃, 60℃, and 70℃) for 15 min in 50 mM Tris-HCl buffer (containing 0.1 mM PLP). The pure enzyme solution was then incubated at different temperatures for 30 min, and the enzyme activity at each temperature was measured to determine the temperature stability of the enzyme. The highest enzyme activity was defined as 100%.

[0084] Specific results are as follows Figure 4 As shown, the optimal temperature of this enzyme is 30℃, and it has good thermal stability, maintaining more than 90% of its enzyme activity at 40℃. The optimal pH of RATA-7 is 9 (the buffer is glycine-sodium hydroxide buffer), and it can tolerate high pH, ​​maintaining more than 80% of its enzyme activity at pH 8-pH 11.

[0085] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An R-type transaminase mutant, characterized in that, Its amino acid sequence is shown in any one of SEQ ID NO.2-SEQ ID NO.

6.

2. The R-type transaminase mutant according to claim 1, characterized in that, The R-type transaminase mutant was obtained by mutating the R-type transaminase with the amino acid sequence shown in SEQ ID NO.

7.

3. The R-type transaminase mutant according to claim 2, characterized in that, The nucleotide sequence of the gene encoding the R-type transaminase is shown in SEQ ID NO.

1.

4. The method for preparing the R-type transaminase mutant according to claim 1, characterized in that, Includes the following steps: S1. Using the plasmid of R-type transaminase with the amino acid sequence shown in SEQ ID NO.7 as a template, the expression plasmid of the R-type transaminase is amplified in its entirety using DNA polymerase to prepare a recombinant expression vector; S2. Transform the recombinant expression vector into host cells to prepare the recombinant expression transformant; S3. Cultivate the recombinant expression transformant and isolate the expressed R-type transaminase mutant.

5. The preparation method according to claim 4, characterized in that, In S1, the sequences of the primers used for amplification are shown in SEQ ID NO.8-SEQ ID NO.

13. Specifically, the R-type transaminase mutant shown in SEQ ID NO.2 was amplified using the primers shown in SEQ ID NO.8-SEQ ID NO.9, the R-type transaminase mutant shown in SEQ ID NO.3 was amplified using the primers shown in SEQ ID NO.10-SEQ ID NO.11, the R-type transaminase mutant shown in SEQ ID NO.4 was amplified using the primers shown in SEQ ID NO.12-SEQ ID NO.13, the R-type transaminase mutant shown in SEQ ID NO.5 was amplified using the primer combination shown in SEQ ID NO.8-SEQ ID NO.11, and the R-type transaminase mutant shown in SEQ ID NO.6 was amplified using the primer combination shown in SEQ ID NO.8-SEQ ID NO.9 and SEQ ID NO.12-SEQ ID NO.

13. In S2, the host cell is Escherichia coli. E. coli BL21(DE3).

6. The preparation method according to claim 4, characterized in that, S3 includes any of the following specific steps: (1) Culture the recombinant expression transformant and isolate the transformant cells containing the R-type transaminase mutant; (2) Cultivate the recombinant expression transformant and isolate the crude enzyme solution containing the R-type transaminase mutant; (3) Cultivate the recombinant expression transformant, isolate the transformant cells containing the R-type transaminase mutant, dry them to obtain R-type transaminase mutant lyophilized bacterial powder, or purify them to obtain R-type transaminase mutant pure enzyme.

7. A catalyst containing a recombinant R-type transaminase mutant, characterized in that, The reorganization R The catalyst for the type 1 transaminase mutant includes any one of the following forms: (1) Transformed cells containing the R-type transaminase mutant of claim 1; (2) A crude enzyme solution containing the R-type transaminase mutant of claim 1; (3) Freeze-dried bacterial powder containing the R-type transaminase mutant of claim 1; (4) A pure enzyme containing the R-type transaminase mutant of claim 1.

8. The use of the R-type transaminase mutant of claim 1 or the recombinant R-type transaminase mutant catalyst of claim 7 in the asymmetric synthesis of (R)-1-(1-naphthyl)-ethylamine.

9. A method for preparing a chiral amine compound, characterized in that, Includes the following steps: Using 1-naphthyl ethyl ketone and isopropylamine as raw materials, and adding the R-type transaminase mutant of claim 1 or the recombinant R-type transaminase mutant catalyst of claim 7 as a catalyst, the reaction is carried out to generate (R)-1-(1-naphthyl)-ethylamine.

10. The preparation method according to claim 9, characterized in that, The preparation method includes the following specific steps: The R-type transaminase mutant catalyst of claim 1 or the recombinant R-type transaminase mutant catalyst of claim 7 is mixed with 1-naphthyl ethyl ketone, acidified isopropylamine, buffer solution and coenzyme PLP to obtain a reaction solution. The reaction solution was placed at 20℃~40℃ and 100 rpm~300 rpm to react and obtain (R)-1-(1-naphthyl)-ethylamine.

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